The human cannabinoid receptor one (CB1) binds ?9-tetrahydrocannabinol, the psychoactive component of Cannabis sativa L., and other cannabimimetic compounds. It is a G-protein coupled receptor (GPCR) that is associated with the central nervous system and exerts its effects primarily via coupling to Gi/Go proteins. The pharmacological effects of CB1 agonists include analgesia, inhibition of nausea, appetite stimulation, antiemetic activity and bronchial dilation while inverse agonists attenuate excessive eating disorders. Any therapeutic strategy targeting CB1 will require that we have a firm understanding of the structural features of the receptor that impact key points in its life cycle: ER integration and cell surface expression, receptor activation, and desensitization and internalization. In Aim 1, we will examine the basis for the weak ER translocation of the CB1 amino terminus (N-tail). The structure of the N-terminus will be determined and accessory proteins and their recognition motifs in the N- and C-terminus will be identified. The role of these motifs in cellular localization including sematodendritic and axonal membrane surface localization in neurons will be examined. In Aim 2, we will build on our identification of structural elements of the receptor critical for distinguishing agonist and inverse agonist interactions and fully map the key contact points in the TM domain and the extracellular region of CB1 that are involved. We will define residues critical for poising the ligand- independent equilibrium of CB1 atypically toward activation and those responsible for the interconversion of this intermediate receptor state to the resting and activated forms of the receptor. In Aim 3, we will take advantage of novel receptor mutants that model different structural states of the receptor to examine linkages between receptor activation, desensitization, and cellular localization. Mutants that model the inactive and active forms of CB1 will provide tools for analyzing the consequences of prolonged treatment with inverse agonists and agonists. We will utilize our expertise in developing structural analyses and binding assays with purified components to examine the molecular basis of these processes with emphasis on the carboxyl terminus (C-tail) of the receptor. In the course of this work we will identify determinants that enhance the cell surface expression of CB1 and strategies for the large-scale preparation of domains of the receptor, and their structural analysis, which could be applied to structural studies of other GPCRs and membrane proteins in general. RELEVANCE: The cannabinoid receptor one (CB1) is a G-protein coupled receptor that is associated with the central nervous system. Research activities that have focused on the role of CB1 in signal transduction have underscored its enormous potential as a target for therapeutic agents. The goal of this project is to understand the structural features that influence key stages in the life cycle of CB1 including cell surface expression, receptor activation, and internalization and ultimately impact its cell surface exposure so that it will be accessible for therapeutic strategies.